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Metodi enzimatici in diagnostica clinica. Enzymatic reaction-based assays. Vantaggi -veloce -molto specifico. Metodi di - Misura in continuo (più facili e più utili) - Misura a tempo fisso. Influenze - pH - temperatura -forza ionica. Michaelis-Menten enzyme kinetics. K m. - PowerPoint PPT Presentation
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Metodi enzimatici in diagnostica clinica
Vantaggi-veloce-molto specifico
Metodi di- Misura in continuo (più facili e più utili)- Misura a tempo fisso
Influenze- pH- temperatura-forza ionica
Enzymatic reaction-based assays
Michaelis-Menten enzyme kinetics
Km
A B CEprim
X Y
Spesso non è possibile un singolo enzima-reazione sequenziale
Eind
t ~ 0 [B] << KmB, vind = Vind.[B]/Km
B - 1st order reaction
t > 0 k.[Eprim] = Vind
vind = Vind.[B]/(KmB + [B]) = Vind/(Km
B/[B] + 1)
[B] 100.KmB - error 1%
It is necessary to use 10 to 100-fold excess of Eind over Eprim!!!
N+
- -N N
NNC
O
NH2
CH2 O P P O
O
O
O
O O
O
OH OH
CH2
O
OH HO
NH2
+
N
- -N N
NN
H+
C
O
NH2
CH2 O P P O
O
O
O
O O
O
OH OH
CH2
O
OH HO
NH2
redox+ 2 [H]- 2 [H]
H
HH
NAD(P)H assay - Wartburg optical test
A + NADH + H+ B + NAD+
Tutte le deidrogenasi NAD o NADP dipendenti utilizzano queso metodo
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Test ottico accoppiato
A + B C + D
C (oD) +NADH E + NAD+
E’ possibile accoppiare una terza reazione
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Calcolo attività enzimatica
• 1 UI =1 micromole di substrato trasformato/min = c / min
• Attività enziamtica (mUI / ml) =
A V 1000
min v d
A = variazione assorbimento a 3401000 = fattore di conversione alle mUIV = volume totale della miscela di reazione = coeff. Estinzione molare di 1 mole/ml di NADH a 340
6.22 cm2/molev = volume del campione (ml)d = cammino ottico (1cm)
NH4+
COO-
C O
CH2
CH2
COO-
COO-
CH NH2
CH2
CH2
COO-
+ NADH + NH4+ + NAD+ + H2O
2-OXOGLUTARATE
GLUTAMATE DEHYDROGENASE
L-GLUTAMATE
CO2 COO-
C O
CH2
P
COO-
C O
CH2
COO-
COO-
C O
CH2
COO-
COO-
CH OH
CH2
COO-
+ CO2 + OH-
PHOSPHOENOLPYRUVATE OXALACETATE
~
PHOSPHOENOLPYRUVATECARBOXYLASE
OXALACETATE
+ NADH + H+
L-MALATE
MALATE DEHYDROGENASE+ NAD+
esempi
NO3- + NADPH + H+ NO2
- + NADP+ + H2O
H2O2 + LEUCODYE DYE + H2O
SO32- + O2 + H2O SO4
2- + H2O2
H2O2 + NADH + H+ 2 H2O + NAD+
NITRATE REDUCTASE
PEROXIDASE
SULFITE OXIDASE
NADH-PEROXIDASE
NO3-
H2 O2
SO32-
Ethanol, Acetaldehyde
ETHANOL + NAD+ACETALDEHYDE + NADH + H+
ACETALDEHYDE + NAD+ + H2O ACETIC ACID + NADH + H+ALDEHYDE DEHYDROGENASE
ALCOHOL DEHYDROGENASE
Acetate
CH2 COO-
COO-
COO-
HO
CH2
ACETATE + ATP + CoA ACETYL-CoA +
ACETYL-CoA + OXALACETATE + H2O
P P
+ CoA
MALATE + NAD+ OXALACETATE + NADH + H+
ACETYL-CoA SYNTHASE
CITRATE
MALATE DEHYDROGENASE
CITRATE SYNTHASE
N N
N
R3
R1 NH
R2
N N
NN
R3
R1 R2
X-
+
+ 2 H+
-2 H+
OO
OH OH
OO
O O
L- ASCORBATE
+ MTT
PMS
+ MTT- FORMAZANE + H+
MTT = 3 -(4,5 -DIMETHYLTHIAZOLYL-2)-2,5- DIPHENYLTETRAZOLIUM BROMIDE
PMS = 5- METHYLPHENAZINIUM BROMIDE
FORMAZANE
BLANK
L-ASCORBATE + O2 ASCORBATE OXIDASE
DEHYDROASCORBATE
+ HX
RED
+ H2O2
C
OH
H
CH2OH
O
O
O2
1
C
OH
H
CH2OH
Ascorbate
578 nm
CITRATE OXALACETATE + ACETATE
PYRUVATE + CO2
OXALACETATE + NADH + H+ L-MALATE + NAD+
PYRUVATE + NADH + H L-LACTATE + NAD+
CITRATE LYASE
MALATE DEHYDROGENASE
L-LACTATE DEHYDROGENASE
L-MALATE + NAD+ OXALACETATE + NADH + H+
OXALACETATE + L-GLUTAMATE L-ASPARTATE + 2-OXOGLUTARATE
MALATE DEHYDROGENASE
ASPARTATE AMINOTRANSFERASE
CH COO-
CH-OOCH2O
FUMARASEL-MALATE+
Citrate
Malate
Fumarate
OXALATEOXALATE DECARBOXYLASE
FORMIATE + CO2
FORMIATE + NAD+ +H2OFORMIATE DEHYDROGENASE
HCO32- + NADH + H+
Oxalate, Formiate
OXALATEOXALATE DECARBOXYLASE
FORMIATE + CO2
FORMIATE + NAD+ +H2OFORMIATE DEHYDROGENASE
HCO32- + NADH + H+
Succinate
Lactate
L-LACTATE + NAD+ PYRUVATE + NADH + H+
PYRUVATE + L-GLUTAMATE L-ALANINE + 2-OXOGLUTARATE
LACTATE DEHYDROGENASE
ALANINE AMINOTRANSFERASE
Pyruvate
PYRUVATE + NADH + H+ L-LACTATE + NAD+L-LACTATE DEHYDROGENASE
2-Oxoglutarate
L-Glutamate
L-Glutamine
2-OXOGLUTARATE + NADH + NH4+
GLUTAMATE DEHYDROGENASE L-GLUTAMATE + NAD+ + H2O
L-GLUTAMINE + H2O GLUTAMINASE
L-GLUTAMATE + NH3
L-GLUTAMATE + NAD+ +H2O 2-OXOGLUTARATE + NADH + NH4+
NADH + IODONITROTETRAZOLIUM CHLORIDE + H+DIAPHORASE
NAD+ + FORMAZAN DYE
GLUTAMATE DEHYDROGENASE
CH2 OH
O
CH2 O-P
O
CH2 O-P
O
O
+ ATP HEXOKINASE+ ADP
-D-GLUCOSE -D-GLUCOSE-6-PHOSPHATE
+ NADP+
GLUCONOLACTON-6-PHOSPHATE
+ NADPH + H+-D-GLUCOSE-6-PHOSPHATE
-D-GLUCOSE-6-PHOSPHATEDEHYDROGENASE
Glucose
SUCROSE + H2OSACCHARASE
-D-GLUCOSE + -D-FRUCTOSE
-D-FRUCTOSE + ATPHEXOKINASE
-D-FRUCTOSE-6-PHOSPHATE + ADP
-D-FRUCTOSE-6-PHOSPHATEPHOSPHOGLUCO ISOMERASE
-D-GLUCOSE-6-PHOSPHATE
Fructose
Sucrose
+ H2O + O2 GLUCOSE OXIDASE
GLUCONOLACTON + H2O2-D-GLUCOSE
CH2
CH
CH2 OH
OH
OHGLYCEROL KINASE
L-GLYCEROL-3-PHOSPHATE + ADP
ADP + PHOSPHOENOLPYRUVATEPYRUVATE KINASE
PYRUVATE + ATP
PYRUVATE + NADH + H+L-LACTATE DEHYDROGENASE
L-LACTATE + NAD+
+ ATP
Glycerol
TRIACYLGLYCEROLS + 3 H2OLIPASE, ESTERASE
GLYCEROL + 3x FATTY ACID
Triacylglycerols
Free fatty acids
FATTY ACID + CoA + ATPACYL-CoA SYNTHASE
AMP + ACYLCoA + P-P
ACYL-CoA + O2ACYL-CoA OXIDASE
ENOYL-CoA + H2O2
H2O2 + 4-AMINOANTIPYRINE + 2,4,6-TRIBROMO-3-HYDROXYBENZOIC ACID
RED DYE + 2 H2O + HBrPEROXIDASE
546 nm
Cholesterol
Bile acids
OH
CHOLESTEROL + O2 4-CHOLESTENON + H2O2
METHANOL + H2O2CATALASE FORMALDEHYDE + 2 H2O
FORMALDEHYDE + NH4+ + 2 ACETYLACETONE 3,5-DIACETYL-1,4-DIHYDROLUTIDINE 3 H2O+
CHOLESTEROL OXIDASE
405 nm
Urea
3--HYDROXY-BILE ACID + NAD+
3--HYDROXYSTEROID DEHYDROGENASE
3-OXO-BILE ACID + NADH + H+
NADH + H+ + NITROBLUE TETRAZOLIUM CHLORIDEDIAPHORASE
NAD+ + FORMAZAN
576 nm
UREA + H2OUREASE
2 NH3 + CO2
2-OXOGLUTARATE + NADH + NH4+
GLUTAMATE DEHYDROGENASEL-GLUTAMATE + NAD+ + H2O
AMP + ATPMYOKINASE
2ADP
ADP + PHOSPHOENOLPYRUVATEPYRUVATE KINASE
L-LACTATE + NAD+PYRUVATE + NADH + H+ L-LACTATE DEHYDROGENASE
ATP + PYRUVATE
AMP/ADP
ATP
GLYCEROL-3-PHOSPHATE + ATP
3-PHOSPHOGLYCERATEKINASE
GLYCERATE-1,3-DIPHOSPHATE + ADP
GLYCERATE-1,3-DIPHOSPHATE + NADH + H+
GLYCERALDEHYDE-3-PHOSPHATEDEHYDROGENASE
GLYCERALDEHYDE-3-PHOSPHATE+ NAD+ + Pi
GLYCERALDEHYDE-3-PHOSPHATETRIOSE PHOSPHATE
ISOMERASEDIHYDROXYACETONE-3-PHOSPHATE
DIHYDROXYACETONE-3-PHOSPHATE + NADH + H+ GLYCEROL-3-PHOSPHATE
DEHYDROGENASE GLYCEROL-3-PHOSPHATE + NAD+
NAD(P)H + H+ + FMN FMN-REDUCTASE NAD(P)+ + FMNH2
LUCIFERASEBACTERIAL
FMN + RCOOH +
FMNH2 + RCHO + O2
NAD(P)H - bioluminescence
ATP - bioluminescence
LUCIFERASEFIREFLY OXYLUCIFERIN + 2 Pi + AMP + CO2 + ATP + D-LUCIFERIN + O2
h. (493 nm)
h. (562 nm)
Metodi in luminescenza
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Diagnostica nell’ischemia cardiaca
L-MALATE + NAD+ OXALACETATE + NADH + H+
OXALACETATE + L-GLUTAMATE L-ASPARTATE + 2-OXOGLUTARATE
MALATE DEHYDROGENASE
ASPARTATE AMINOTRANSFERASE
Transaminasi glutamico ossalacetica (GOT)
GOT aumenta dopo 6 ore l’infarto
max a 36 ore
livelli normali 5gg
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
• Creatina chinasiCH2 OH
O
CH2 O-P
O
CH2 O-P
O
O
+ ATP HEXOKINASE+ ADP
-D-GLUCOSE -D-GLUCOSE-6-PHOSPHATE
+ NADP+
GLUCONOLACTON-6-PHOSPHATE
+ NADPH + H+-D-GLUCOSE-6-PHOSPHATE
-D-GLUCOSE-6-PHOSPHATEDEHYDROGENASE
Fosfocreatina + ADP creatina + ATP
Aumenta dopo 3 ore dall’infarto max 24-30 h
livelli normali 3gg
Creatina fosfato
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
L-LACTATE + NAD+ PYRUVATE + NADH + H+
PYRUVATE + L-GLUTAMATE L-ALANINE + 2-OXOGLUTARATE
LACTATE DEHYDROGENASE
ALANINE AMINOTRANSFERASE
Lattico deidrogenasi
Aumento entro 2 ore max 60 ore
ritorno alla norma lento
Troponine cardiache:molto specifiche per il cuore
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Dosaggio del substrato• 10 ml di NADH 20 mM (Ci) vengono messi in 2 ml di soluzione tampone
(CfNADH = 100 µM)• 10 ml di piruvato prelevati dal liquido biologico (siero)• 10 ml di lattico deidrogenasi
• Piruvato + NADH + H+ lattato + NAD+
• Sapendo che l’e molare di NADH è 6,22 mM-1 cm-1• 0,1 mM di NADH avrà un assorbanza di 0,622 (Ai)• Nella lettura Af ottengo: 0,322 A = lC A = 0,622 – 0,322 = 0.3 C = A/l = 0.3 / 6,22 mM-1 cm-1 x cm = 0,48 mM = 48 mM• Le micromoli di piruvato nei 2 ml della cuvetta saranno uguali a• 48 mM x 2 ml / 1000 = 0, 096 mmoli• [piruvato] = 0,096 / 10 ml (volume usato) = 9,6 mM
(concentrazione del piruvato)•